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Team:UNITN-Trento/Notebook/Labposts/07/57 - Revision history
2024-03-28T14:13:15Z
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Ggirelli at 10:45, 3 October 2013
2013-10-03T10:45:57Z
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{"date" : "2013-07-<ins class="diffchange diffchange-inline">24</ins>","author" : "<ins class="diffchange diffchange-inline">gabriele</ins>","title" : "<ins class="diffchange diffchange-inline">Inocula</ins>","content" : "<html>I <ins class="diffchange diffchange-inline">checked </ins>the <ins class="diffchange diffchange-inline">plates prepared by Caterina <a href=\"https://2013</ins>.<ins class="diffchange diffchange-inline">igem</ins>.<ins class="diffchange diffchange-inline">org</ins>/<ins class="diffchange diffchange-inline">wiki/index.php?title=</ins>Team:UNITN-Trento/<ins class="diffchange diffchange-inline">Notebook#tn-post-2013-07-23-caterina\">yesterday<</ins>/<ins class="diffchange diffchange-inline">a>.</ins><<ins class="diffchange diffchange-inline">br/</ins>><center><<ins class="diffchange diffchange-inline">table class</ins>=\"<ins class="diffchange diffchange-inline">tn-sp-table\"><tr><th>plate<</ins>/<ins class="diffchange diffchange-inline">th><th>#colonies<</ins>/<ins class="diffchange diffchange-inline">th><th>#inocula<</ins>/<ins class="diffchange diffchange-inline">th><</ins>/<ins class="diffchange diffchange-inline">tr><tr><td>ON Ctrl<</ins>/<ins class="diffchange diffchange-inline">td><td>0</td><td>0</td></tr><tr><td>ON 1:1</td><td>6</td><td></ins>5<ins class="diffchange diffchange-inline"><</ins>/<ins class="diffchange diffchange-inline">td><</ins>/<ins class="diffchange diffchange-inline">tr><tr><td>ON 1</ins>:<ins class="diffchange diffchange-inline">3</td</ins>><ins class="diffchange diffchange-inline"><td>0</ins></<ins class="diffchange diffchange-inline">td</ins>><ins class="diffchange diffchange-inline"><td>0</ins></<ins class="diffchange diffchange-inline">td</ins>><<ins class="diffchange diffchange-inline">/tr><tr><td>Short Ctrl</td><td>0</td><td>0</td></tr><tr><td>Short 1:1</td><td>1</td><td>1</td></tr><tr><td>Short 1:3</td><td>1</td><td>1</td></tr></table></center><br/</ins>>As you <ins class="diffchange diffchange-inline">might notice, an O/N 1:3 ligation was never performed, </ins>the <ins class="diffchange diffchange-inline">problem is that Caterina wrote the wrong labels on the plates</ins>, <ins class="diffchange diffchange-inline">so we can't link </ins>the <ins class="diffchange diffchange-inline">plates back to the ligations</ins>.<ins class="diffchange diffchange-inline">.. anyway, we will keep the denomination </ins>of <ins class="diffchange diffchange-inline">the plates from now onward</ins>.</html>","tags" : "<ins class="diffchange diffchange-inline">SAMsynthetase</ins>"}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"date" : "2013-07-<del class="diffchange diffchange-inline">27</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"author" : "<del class="diffchange diffchange-inline">thomas</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"title" : "<del class="diffchange diffchange-inline">AraCpBAD + EFE + Venus transformation</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"content" : "<html><del class="diffchange diffchange-inline">Once </del>I <del class="diffchange diffchange-inline">confirmed </del>the <del class="diffchange diffchange-inline">construct through an electrophoresis analisis, I decided to transorm the sample C into NEB10b cells</del>. <del class="diffchange diffchange-inline">I plated then 100 ul of cells and putted the plate in static incubation at 37&deg;C overnight</del>.<del class="diffchange diffchange-inline"><</del>/<del class="diffchange diffchange-inline">html>{{:</del>Team:UNITN-Trento/<del class="diffchange diffchange-inline">Templates/Styles</del>/<del class="diffchange diffchange-inline">Spoiler|Plate Image|</del><<del class="diffchange diffchange-inline">html</del>><center><<del class="diffchange diffchange-inline">img src</del>=\"<del class="diffchange diffchange-inline">https:</del>//<del class="diffchange diffchange-inline">static.igem.org</del>/<del class="diffchange diffchange-inline">mediawiki</del>/<del class="diffchange diffchange-inline">2013</del>/5/<del class="diffchange diffchange-inline">53</del>/<del class="diffchange diffchange-inline">Tn-2013_efe%2Bvenus_plate.JPG\" style =\"width</del>: <del class="diffchange diffchange-inline">450px\"</del>></<del class="diffchange diffchange-inline">center</del>></<del class="diffchange diffchange-inline">html</del>><del class="diffchange diffchange-inline">}}</del><<del class="diffchange diffchange-inline">html</del>> As you <del class="diffchange diffchange-inline">can see from </del>the <del class="diffchange diffchange-inline">picture</del>, the <del class="diffchange diffchange-inline">colonies grown</del>. <del class="diffchange diffchange-inline">One </del>of <del class="diffchange diffchange-inline">them was then picked up and inoculated overnight</del>.</html>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"tags" : "<del class="diffchange diffchange-inline">EFE-Venus</del>"</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>}</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/57&diff=273446&oldid=prev
Ggirelli at 08:44, 3 October 2013
2013-10-03T08:44:46Z
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<td colspan='2' style="background-color: white; color:black;">Revision as of 08:44, 3 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "date" : "2013-07-<del class="diffchange diffchange-inline">11</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "date" : "2013-07-<ins class="diffchange diffchange-inline">27</ins>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "author" : "<del class="diffchange diffchange-inline">gabriele</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "author" : "<ins class="diffchange diffchange-inline">thomas</ins>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "title" : "<del class="diffchange diffchange-inline">Extraction (again) and ligation</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "title" : "<ins class="diffchange diffchange-inline">AraCpBAD + EFE + Venus transformation</ins>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "content" : "<html><del class="diffchange diffchange-inline"><h3>SAM synthetase extraction</h3><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-10-gabriele-viola\">Last time</a> </del>I <del class="diffchange diffchange-inline">determined </del>the <del class="diffchange diffchange-inline">correct protocol </del>to <del class="diffchange diffchange-inline">extract SAM synthetase from </del>the <del class="diffchange diffchange-inline"><i>E. coli</i> using the new plasmid (at the end, the protocol was <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">the usual one</a>)</del>. I <del class="diffchange diffchange-inline">performed three PCRs (G1, G2, G3) </del>and <del class="diffchange diffchange-inline">run </del>the <del class="diffchange diffchange-inline">products on 1% agarose gel</del>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|<del class="diffchange diffchange-inline">Gel</del>|<html><center<del class="diffchange diffchange-inline">><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>G2</td><td>G3</td></tr></table</del>><img src=\"https://static.igem.org/mediawiki/2013/<del class="diffchange diffchange-inline">f</del>/<del class="diffchange diffchange-inline">f4</del>/Tn-<del class="diffchange diffchange-inline">20130711-GG_SAMsynthetase_extraction_EXSP</del>.<del class="diffchange diffchange-inline">jpg</del>\" <del class="diffchange diffchange-inline">alt</del>=<del class="diffchange diffchange-inline">\"Gel\" title=\"Gel</del>\" width<del class="diffchange diffchange-inline">=\"</del>450px\" <del class="diffchange diffchange-inline">/</del>></center></html>}}<html><del class="diffchange diffchange-inline"><br><hr><h3>Ligation</h3>At first</del>, <del class="diffchange diffchange-inline">I added 1&micro;l of SAP to pSB1C3 O/N digestion and 1&micro;l of DpnI to SAMsynthetase O/N digestion, and incubated </del>the <del class="diffchange diffchange-inline">samples at 37&deg;C for 1</del>.<del class="diffchange diffchange-inline">5h. Then I quantified the digestions.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>SAM synthetase</td><td>12.2ng/&micro;l</td></tr><tr><td>pSB1C3</td><th>14.0ng/&micro;l</th></tr></table></center><br/>Then I performed the ligation </del>and <del class="diffchange diffchange-inline">left the reaction run for 2h at room temperature</del>.<del class="diffchange diffchange-inline"></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>CTRL</th><th>1:1</th><th>1:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">4&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>9.15&micro;l</td><td>18.3&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>20.71&micro;l</td><td>11.56&micro;l</td><td>2.41&micro;l</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10&beta;</del></html>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "content" : "<html><ins class="diffchange diffchange-inline">Once </ins>I <ins class="diffchange diffchange-inline">confirmed </ins>the <ins class="diffchange diffchange-inline">construct through an electrophoresis analisis, I decided </ins>to <ins class="diffchange diffchange-inline">transorm </ins>the <ins class="diffchange diffchange-inline">sample C into NEB10b cells</ins>. I <ins class="diffchange diffchange-inline">plated then 100 ul of cells </ins>and <ins class="diffchange diffchange-inline">putted </ins>the <ins class="diffchange diffchange-inline">plate in static incubation at 37&deg;C overnight</ins>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|<ins class="diffchange diffchange-inline">Plate Image</ins>|<html><center><img src=\"https://static.igem.org/mediawiki/2013/<ins class="diffchange diffchange-inline">5</ins>/<ins class="diffchange diffchange-inline">53</ins>/Tn-<ins class="diffchange diffchange-inline">2013_efe%2Bvenus_plate</ins>.<ins class="diffchange diffchange-inline">JPG</ins>\" <ins class="diffchange diffchange-inline">style </ins>=\"width<ins class="diffchange diffchange-inline">: </ins>450px\"></center></html>}}<html> <ins class="diffchange diffchange-inline">As you can see from the picture</ins>, the <ins class="diffchange diffchange-inline">colonies grown</ins>. <ins class="diffchange diffchange-inline">One of them was then picked up </ins>and <ins class="diffchange diffchange-inline">inoculated overnight</ins>.</html>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "tags" : "<del class="diffchange diffchange-inline">SAMsynthetase</del>"</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "tags" : "<ins class="diffchange diffchange-inline">EFE-Venus</ins>"</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>}</div></td></tr>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/57&diff=110481&oldid=prev
Ggirelli: moved Team:UNITN-Trento/Notebook/Labposts/07/70 to Team:UNITN-Trento/Notebook/Labposts/07/57
2013-09-09T07:55:50Z
<p>moved <a href="/Team:UNITN-Trento/Notebook/Labposts/07/70" title="Team:UNITN-Trento/Notebook/Labposts/07/70">Team:UNITN-Trento/Notebook/Labposts/07/70</a> to <a href="/Team:UNITN-Trento/Notebook/Labposts/07/57" title="Team:UNITN-Trento/Notebook/Labposts/07/57">Team:UNITN-Trento/Notebook/Labposts/07/57</a></p>
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<td colspan='1' style="background-color: white; color:black;">Revision as of 07:55, 9 September 2013</td>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/57&diff=110476&oldid=prev
Ggirelli: moved Team:UNITN-Trento/Notebook/Labposts/90 to Team:UNITN-Trento/Notebook/Labposts/07/70
2013-09-09T07:55:23Z
<p>moved <a href="/Team:UNITN-Trento/Notebook/Labposts/90" class="mw-redirect" title="Team:UNITN-Trento/Notebook/Labposts/90">Team:UNITN-Trento/Notebook/Labposts/90</a> to <a href="/Team:UNITN-Trento/Notebook/Labposts/07/70" title="Team:UNITN-Trento/Notebook/Labposts/07/70">Team:UNITN-Trento/Notebook/Labposts/07/70</a></p>
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<td colspan='1' style="background-color: white; color:black;">Revision as of 07:55, 9 September 2013</td>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/57&diff=78565&oldid=prev
Ggirelli: Created page with "{ "date" : "2013-07-11", "author" : "gabriele", "title" : "Extraction (again) and ligation", "content" : "<html><h3>SAM synthetase extraction</h3><a href=\"http://2013.igem.o..."
2013-08-19T13:35:12Z
<p>Created page with "{ "date" : "2013-07-11", "author" : "gabriele", "title" : "Extraction (again) and ligation", "content" : "<html><h3>SAM synthetase extraction</h3><a href=\"http://2013.igem.o..."</p>
<p><b>New page</b></p><div>{<br />
"date" : "2013-07-11",<br />
"author" : "gabriele",<br />
"title" : "Extraction (again) and ligation",<br />
"content" : "<html><h3>SAM synthetase extraction</h3><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-10-gabriele-viola\">Last time</a> I determined the correct protocol to extract SAM synthetase from the <i>E. coli</i> using the new plasmid (at the end, the protocol was <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">the usual one</a>). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>G2</td><td>G3</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/f/f4/Tn-20130711-GG_SAMsynthetase_extraction_EXSP.jpg\" alt=\"Gel\" title=\"Gel\" width=\"450px\" /></center></html>}}<html><br><hr><h3>Ligation</h3>At first, I added 1&micro;l of SAP to pSB1C3 O/N digestion and 1&micro;l of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37&deg;C for 1.5h. Then I quantified the digestions.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>SAM synthetase</td><td>12.2ng/&micro;l</td></tr><tr><td>pSB1C3</td><th>14.0ng/&micro;l</th></tr></table></center><br/>Then I performed the ligation and left the reaction run for 2h at room temperature.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>CTRL</th><th>1:1</th><th>1:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">4&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>9.15&micro;l</td><td>18.3&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>20.71&micro;l</td><td>11.56&micro;l</td><td>2.41&micro;l</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10&beta;</html>",<br />
"tags" : "SAMsynthetase"<br />
}</div>
Ggirelli